Formulations of therapeutically effective proteins, such as G-CSF, remain difficult to formulate for extended shelf life in vitro and in vivo activity. Formulations of such proteins must maintain their activity and biological integrity for appropriate periods of time for effective treatment. In addition, formulations of such proteins must be manufacturable as well as capable of being administered to an animal in a pharmaceutically acceptable manner.
Pharmaceutical compositions of proteins have been provided in frozen or lyophilized form and maintained in vitro under storage conditions, which maintain protein activity for extended periods of time. Lyophilized preparations are reconstituted prior to use with pharmaceutically acceptable diluents, such as sterile water for injection. Pharmaceutical compositions of proteins have also been provided in liquid form. Such liquid protein formulations are difficult to maintain in storage due to the loss of protein activity over time, particularly at elevated temperatures.
Formulations of therapeutically effective proteins, whether in solid (lyophilized) or liquid form, are difficult to administer to animals without sudden loss of activity after administration, such as by subcutaneous injection, to the animal. Rapid loss of protein activity at the injection site renders the protein inconvenient for treating infections in mammals since effective therapy requires daily doses during desired periods of coverage. Granulocyte colony stimulating factors (G-CSFs), such as bovine granulocyte colony stimulating factor (bG-CSF), are unstable at or above 40° C. due to loss in secondary structure and disulfide interchange and subsequent loss in activity. This loss in activity occurs at the injection site since bovine body temperature is around 40° C. and the injection site is at physiological pH range.
Various protein formulations for extending shelf life are known. U.S. Pat. No. 5,104,651, issued Apr. 14, 1992 (Boone et al.), refers to a pharmaceutical composition of G-CSF and an acid at a pH in the range of 3.0–3.7 with a conductivity of less than 1000 μmhos/cm. U.S. Pat. No. 4,992,271, issued Feb. 12, 1991 (Fernandes et al.), refers to a pharmaceutical composition containing a biologically active recombinant interleukin 2 protein dissolved in an aqueous based carrier medium at a pH of 6.8 to 7.8 and which further contains a stabilizer for the protein, such as human serum albumin. U.S. Pat. No. 4,623,717, issued Nov. 18, 1986 (Fernandes et al.), refers to pasteurized therapeutically active protein compositions whereby thermally sensitive, therapeutically active proteins are pasteurized by mixing the protein with a stabilizing amount of a sugar or reduced sugar and an amino acid prior to pasteurization. U.S. Pat. No. 4,645,830, issued Feb. 24, 1987 (Yasushi et al.), refers to a stable interleukin 2 composition containing interleukin 2, human serum albumin and a reducing compound, at a pH of 3 to 6 in solution. U.S. Pat. No. 4,647,454, issued Mar. 3, 1987, (Cymbalista), refers to a method of stabilizing human fibroblast interferon with polyvinyl pyrrolidone. U.S. Pat. No. 4,675,184, issued Jun. 23, 1987 (Hasegawa et al.), refers to a pharmaceutical composition for treating viral infections containing interferon, a tri or higher polyhydric sugar alcohol, an organic buffer and a pharmaceutical carrier or diluent, wherein the composition has a pH of about 3 to 6. All of the aforementioned references are incorporated by reference in their entirety.
One example of a therapeutically effective class of proteins is that of granulocyte colony stimulating factors (G-CSFs). Granulocyte colony stimulating factor (G-CSF) is one of several glycoprotein growth factors known as colony stimulating factors. Such colony stimulating factors support the proliferation of haemopoietic progenitor cells and stimulate proliferation of specific bone marrow precursor cells and their differentiation into granulocytes. In addition, G-CSF is capable of stimulating neutrophilic granulocyte colony formation and to inducing terminal differentiation of murine myelomonocytic leukemic cells in vitro. G-CSF has also been shown to stimulate the functional activities of neutrophils resulting in enhanced microbiocidal activity. G-CSF has a known amino acid sequence of 174 amino acids.
Recombinant forms of CSFs and G-CSFs have been prepared. The cloning and expression of DNA encoding for human G-CSF is known (Nagata, S. et al., Nature, 319, 415–418 (1986). WO-A-8604606 and WO-A-8604506 describe a gene encoding human G-CSF. U.S. Pat. No. 5,606,024, issued Feb. 25, 1997 (Boone et al.) and U.S. Pat. No. 5,472,857 issued Dec. 5, 1995, describe the DNA sequence encoding canine granulocyte colony stimulating factor (cG-CSF) as well as a method for treating or preventing infections in canine or feline animals by administering effective amounts of human and canine G-CSF to such animals. U.S. Pat. No. 4,810,643, issued Mar. 7, 1989 (Souza), describes human G-CSF like polypeptides. European Patent Application No. 719 860, published Jul. 3, 1996, describes the amino acid sequence of naturally occurring bovine granulocyte colony stimulating factor (bG-CSF), the DNA sequence encoding for bG-CSF and a method for treating or preventing mastitis in an animal by administering to the animal an effective amount of G-CSF. WO-A-8702060 describes human G-CSF like polypeptide, sequences encoding them and methods of producing them. U.S. Pat. No. 4,833,127, issued May 23, 1989 (Ono et al.), describes a novel biologically active human granulocyte colony stimulating factor. European Patent Application No. 612 846, published Aug. 31, 1994, describes certain G-CSF analogs and compositions containing such analogs. All of the aforementioned references are incorporated by reference in their entirety.
Granulocyte colony stimulating factors are useful as anti-infective agents which increases the immune competence of the animal rather than targeting a specific microbial target necessary for growth or virulence. There are few other commercially available agents used in veterinary medicine that target non-specific immune responses leading to increased resistance to microbial infection. Available control measures are limited to conventional antimicrobials and a limited number of biologicals. Economic losses associated with milk withdrawal periods in cattle limit the utility of conventional antimicrobials. Current vaccines target a limited number of species and the field efficacy of these agents vary widely. The most successful vaccines, (E. coli J5) are limited in their world wide use due to safety concerns associated with endotoxin contamination.
Mastitis is a major disease problem affecting dairy producers worldwide. Economic losses in the United States associated with mastitis exceed $1 billion annually. These losses are associated with mortalities, milk discard, acute and chronic decreases in milk production, increased early culling and drug and veterinary labor expenses. Periparturient dairy cows exhibit impaired immune responsiveness (neutrophil function) which increase their susceptibility to bacterial infections of the mammary gland. The impact of this increased susceptibility is exemplified by the fact that about 40% of new clinical intramammary infections occur within the first two weeks after calving. Mastitis is associated with a wide variety of bacterial pathogens including both Gram positive and Gram negative organisms. Some of the known pathogenic microorganisms causing mastitis are Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, Aerobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa. These pathogens enter the udder through the test canal and produce inflammation of the milk producing tissue causing the formation of scar tissue, which can result in a permanent loss of milk producing capacity. Various forms of mastitis include: udder infection, chronic mastitis, clinical mastitis and subclinical mastitis.
Current antimicrobial therapies and vaccines possess a number of deficiencies that limit their utility in lactating cows. Antibiotic therapy to control mastitis has been found lacking. There is a need for a biotherapeutic agent, which is useful in restoring normal immune competence resulting in the decreased incidence and severity of mastitis.
Bovine respiratory disease, also referred to as shipping fever, is another common disease affecting cattle. Bovine respiratory disease affects cattle after shipment either into feedlots or onto pasture and results from a variety of stresses affecting cattle including weaning, castration, dehorning, fasting, overcrowding, exposure to infectious agents, diet changes and temperature changes, in combination with infection by any of several known pathogens. Pasteurella haemolytica is one such common pathogen resulting in damage to the respiratory system of cattle.
Several additional infectious diseases, including various reproductive diseases, are also known to affect humans, swine, cattle, dogs, cats, horses, goats and sheep. One example of such a disease, occurring in cattle, is metritis.
There is a need for a stable protein composition which remains therapeutically effective for sustained periods of time in vivo. In addition, there is a need for formulations of proteins that provide for extended in vitro shelf life and storage.